Our proposed research underscores several major concepts such as the issue of rickettsial virulence, rickettsial fitness in diverse vertebrate and invertebrate hosts, and molecular dynamics of pathogen-host (flea/rat) interactions. In this competing renewal application, we build upon recent accomplishments, and now focus on dissecting the molecular correlates that define the murine typhus zoonosis. Thus, we will use an integrated approach including the combination of rickettsial genome data mining, pathogen phylogenomic analysis and empirical studies to investigate the patterns of rickettsial and host transcriptome profiles and to define whether host-association directs the degree of rickettsial virulence. The aims of this competitive renewal is to (1) determine if flea immune response proteins limit the growth of R. typhi (Aim I) and (2) functionally characterize a set of rickettsial genes that were identified as having a potential role in virulence (Aim II). The specific aims of the proposal are designed to highlight the strategies employed by both the host and the Rickettsia typhi to ensure their own survival. Under the aim I, we will test the hypothesis that flea immune responsive proteins limit R. typhi growth. To implicate an interaction between R. typhi and each immune responsive protein in vivo, immunological, biochemical and molecular approaches will be used to demonstrate colocalization and anti-rickettsial properties of each immune responsive protein. Additionally, RNAi will be used to determine if suppression of immune gene expression affects rickettsial growth in vivo. Under the second aim we will test the hypothesis that secretory proteins play a cardinal role in rickettsial virulence. In order to assess this hypothesis, we will utilize an integrated approach that includes a genome-wide identification and molecular analysis of R. typhi secretory proteins and functional analysis of selected potential virulence genes by anti-sense RNA technology. PROJECT NARRATIVE: Murine typhus is a good example of a disease whose importance is not adequately appreciated except by the patient, and, even today, in most parts of the world, he will never know what ails him because the diagnosis will not be made. An intimate association between the causative agent of murine (endemic) typhus, Rickettsia typhi, rats and their fleas underlie the expansion of this disease into rural communities as the urbanization of rural settings is on the increase. Thus, R. typhi not only re-emerge in many coastal regions of the world as a result of relaxed hygiene but also become an emerging pathogen in rural animal populations. Our proposed research underscores several major concepts in rickettsial virulence and pathogenesis by using murine typhus animal model. Our research advances the understanding the biology of R. typhi by providing information on pathogen gene products that could be used in developing better rickettsial vaccines and/or therapeutics.